Enhanced production of antibodies utilizing insolubilized immune complexes

ABSTRACT

A method for enhancing production of antibodies through immunization with insolubilized immune complexes is disclosed. Purified antigen or heterogeneous antigen mixtures may be combined with polyclonal or monoclonal antibody and the resultant complex bound to an insolubilized matrix to form insolubilized immune complexes. Methods for improving the immunogenicity of a soluble antigen and for producing monoclonal anti-iodiotypic antibodies are also disclosed. Monoclonal antibodies that are specific for a distinct, as yet unrecognized epitope may be produced by another disclosed method . Insolubilized immune complexes, comprising antigen and antibody that is ether directly linked to Sepharose® or absorbed onto insolubilized protein A, and immunosorbents, comprising antibody absorbed onto insolubilized protein A, are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 07/391,286, filed Aug. 8, 1989, now abandoned,which is a continuation-in-part application of allowed U.S. patentapplication Ser. No. 024,632, filed Mar. 11, 1987, which issued as U.S.Pat. No. 4,879,225, which is a continuation-in-part application of U.S.patent application Ser. No. 876,828, filed on June 20, 1986, nowabandoned.

TECHNICAL FIELD

The present invention is for a method of enhanced production ofantibodies to a variety of antigens utilizing insoluble immunecomplexes. Purified antigen, enriched antigen present with othercontaminating antigens, and heterogeneous mixtures are employed assources of antigen. Polyclonal monospecific, polyclonal polyspecific, ormonoclonal antibodies are combined with antigen and a particulate,insoluble carrier, such as protein A-Sepharose® or methacrylate beads,to form insolubilized immune complexes. Antibodies to the antigencomponent of such complexes are produced with high efficiency, and insome cases, with improved specificity over original antisera ormonoclonal antibodies.

BACKGROUND ART

A variety of protocols in the prior art have been employed forproduction of monoclonal and polyclonal antibodies to glycoproteins, toglycolipids and to tumor-associated antigens. For example, production ofmonoclonal antibodies can utilize whole cells, which are a highlyimmunogenic vehicle. This method, however, is fraught with the problemof a low efficiency in producing antibodies of interest. In addition,purified, soluble antigens are poorly immunogenic in mice, in contrastto the same immunogens in other species such as rabbits and sheep.

Presentation of an antigen in an insoluble form may improveimmunogenicity of soluble antigen preparations in mice. For example,alum precipitation of carcinoembryonic antigen in some cases enhancesimmunogenicity. However, adjuvants are often required to achieve desiredlevels of antibody production. Traditionally, adjuvants, such asFreund's, have been admixed with soluble antigen preparations forimmunizing rabbits or goats. However, adjuvant is oftentimes ineffectivein mice; even though it may produce detectable titers of antibody, theyield of antigen-specific hybridomas is low. The present inventiondiscloses an insolubilized immunogen that is effective in producingantigen-specific hybridomas.

Soluble immunogen preparations described in the prior art also usuallyrequire a great deal of effort for isolation of sufficient quantities ofpurified antigen. Most protocols for immunizing with soluble antigensutilize 50-100 μg of purified antigen. Preparing this dosage of antigeninvolves considerable effort in biochemical purification, because mostcell surface or cytoplasmic antigens are produced only in small amountsby cells. An advantage of the present invention is that it provides aprocess for enrichment of a given antigen from a heterogeneous mixture,thus reducing the need for purification. The term "enrichment," as usedherein, means an increased concentration of the antibody or antigen ofinterest.

Another advantage provided by this invention is the production ofmonoclonal antibodies to epitopes on the same antigen that ar distinctfrom the epitope identified by the antibody used in preparation of theimmunogen. The need for antibodies to multiple epitopes of an antigenhas been previously demonstrated. For example, it has been shown thatmore than one epitope can exist within a glycoprotein species, and thatdifferent combinations of epitopes can be found on differentsubpopulations of glycoprotein molecules. Thus, not all epitopes have tobe on all the glycoprotein molecules and/or have the same distributionwithin a given tissue. More specifically, the inventors have found thatthe degree of antigenic heterogeneity in a human melanoma-associatedantigen system is dependent upon the epitope recognized by themonoclonal antibody (Morgan et al., Mol. Immunol. 23:193-200, 1986). Onemonoclonal antibody (MAb), that recognized an epitope displayed on mostof the melanoma glycoprotein molecules, had less antigenic heterogeneitythan a MAb that recognized an epitope present only on a subpopulation ofglycoprotein molecules. Utilizing monoclonal antibodies tocarcinoembryonic antigen (CEA), the CEA preparation can be subdividedinto populations of molecules bearing unique epitopes. This also hasbeen demonstrated with monoclonal antibodies to Class IIhistocompatibility antigens (HLA-DR antigens). Thus, the generation ofunique antibodies to specific epitopes on a given antigen is useful forthe optimization of therapeutic applications of monoclonal antibodiesinvolving delivery of radioisotopes, drugs, or toxins to a target tumor.

As with antigen purification, the prior art method of producingmonoclonal antibodies to specific epitopes of a given antigen islaborious. The prior art methodology involves immunization with solublepurified antigen isolated by a series of steps: production of largequantities of a given monoclonal antibody, purification of thatantibody, insolubilization of that antibody on an insoluble matrix(e.g., Sepharose®, an agarose gel, optionally cross-linked, inbead-form), and typically utilization of at least one otherchromatography procedure (e.g., lectin affinity chromatography, ionexchange chromatography, gel filtration or hydrophobic chromatography)in combination with monoclonal antibody affinity chromatography topurify the antigen from a heterogeneous antigen mixture. This method isexpensive and time-consuming, and requires large numbers of cells as thesource of the impure antigen. In addition, once isolated, the solubleantigen preparation is poorly immunogenic in mice. Thus, there is a needin the art for more efficient and less time-consuming methods ofgenerating monoclonal antibodies to epitopes of antigens distinct fromthose recognized by existing monoclonal antibodies. Within the presentinvention, the source of antigen need not be purified, and yetimmunogenicity is enhanced.

There is also a need in the art for a method of producing monoclonalantibodies to antigens for which polyclonal antisera exist. If thesepolyclonal antisera do exist, although in small amounts, in low titers,or in polyspecific form (containing many antibodies of differentspecificities), they may be used in the present invention for productionof monoclonal antibodies. In many cases, the amounts of these antiseraare limited, are insufficient for antibody affinity chromatography, andare thus useless for immunization procedures involving soluble purifiedantigen. An additional benefit of the immunization process of thepresent invention is the ability to generate monoclonal antibodies ofdifferent specificity compared to the polyclonal antiserum used in theimmunization.

There is also a need in the art for a method for efficiently producingmonoclonal anti-idiotypic antibodies, which recognize theantigen-combining site of a monoclonal antibody (MAb) of murine origin.It has been postulated and subsequently verified in certain animalmodels that anti-idiotypic antibodies can act to regulate the immuneresponse in both a positive and a negative manner. In certain instances,anti-idiotypic antibodies can substitute for purified antigen in theproduction of vaccines, thus eliminating the need for biochemicalpurification of a given antigen.

As shown in certain animal models, anti-idiotypic antibodies utilized asvaccines may prove useful in adjuvant therapy of cancer. The field ishandicapped by difficulties in producing monoclonal antibodies of murineorigin to other murine monoclonal antibodies. Murine MAbs to human andrabbit antibodies have been successfully produced. However, most MAbs totumor-associated antigens are of mouse origin. The combining site ofthese tumorspecific murine MAbs may be poorly immunogenic, presumablydue to self-tolerance induced by common sequences within the Fc region.It appears that species distinction within the Fc region may result inenhanced immunogenicity of the antigen combining site (idiotope). In thepresent invention, the use of an immunosorbent enhances the generationof antibodies to the idiotope of a monoclonal antibody.

Another advantage of the present invention is that it provides anefficient technique for generating a battery of monoclonal antibodiesthat are directed against multiple epitopes of a given antigen. Thismethodology facilitates the production of monoclonal antibodies thatrecognize immunorecessive epitopes of an antigen. As a result, a varietyof monoclonal antibodies may be bound to one target cell or antigen,thereby enhancing the delivery of a higher dose of conjugated toxin orradioisotope to a target site. Delivery of an increased concentration ofimmunoconjugates improves cytotoxicity or imaging properties, becausemultiple distinct epitopes of a target cell can be bound. In addition,the availability of a multiplicity of monoclonal antibodies will permittargeting of a variety of epitopes, some of which may be unique to aparticular target cell. This unique binding capability may allowselection of monoclonal antibodies that exhibit little or nocross-reactivity with epitopes of normal cells, which in turn mightpermit administration of higher doses of immunoconjugates.

DISCLOSURE OF THE INVENTION

The present invention discloses a method for enhancing production ofantibodies, comprising the steps of absorbing antibody ontoinsolubilized protein A, thereby forming an immunosorbent; combining anantigen with the immunosorbent, forming an insolubulized immune complex;immunizing an animal with the insolubilized immune complex; andcollecting antiserum from the immunized animal.

A related aspect of the present invention discloses a method forimproving the immunogenicity of a soluble antigen, thereby enhancingproduction of specific monoclonal antibodies, comprising the steps ofabsorbing antibody onto insolubilized protein A, thereby forming animmunosorbent; combining the soluble antigen with the immunosorbent,forming an insolubilized immune complex; immunizing an animal with theinsolubilized immune complex; fusing spleen cells from the immunizedanimal to myeloma cells to form hybridomas capable of producingmonoclonal antibodies to the soluble antigen; culturing the hybridomasto produce the monoclonal antibodies: and collecting the monoclonalantibodies as a product of the hybridomas.

A further aspect of the present invention discloses a method forproducing monoclonal antibodies specific for an epitope distinct fromthat recognized by an existing monoclonal antibody, comprising the stepsof absorbing an existing monoclonal antibody directed against a firstepitope of a polyvalent antigen onto insolubilized protein A, therebyforming an immunosorbent; combining the immunosorbent with the antigen,forming an insolubilized immune complex wherein the first epitope ismasked by the existing monoclonal antibody; immunizing an animal withthe insolubilized immune complex; fusing spleen cells from the immunizedanimal to myeloma cells to form hybridomas capable of producing secondmonoclonal antibodies to the particular epitope; culturing thehybridomas to produce the second monoclonal antibodies specific for theparticular epitope; and collecting the second monoclonal antibodies as aproduct of the hybridomas.

Another aspect of the present invention discloses a method for producingmonoclonal anti-idiotypic antibodies, comprising the steps of absorbinga first antibody onto insolubilized protein A, thereby forming animmunosorbent; immunizing an animal with the immunosorbent; fusingspleen cells from the immunized animal to myeloma cells to formhybridomas capable of producing monoclonal antibodies to an idiotope ofthe first antibody; culturing the hybridomas to produce the monoclonalantibodies; and collecting the monoclonal antibodies as a product of thehybridomas.

Insolubilized immune complexes for use as immunogens and immunosorbentsfor use in producing monoclonal anti-idiotypic antibodies are alsodisclosed. In addition, a variety of monoclonal antibodies that bind tomultiple epitopes of a tumor-associated antigen are disclosed.

A further aspect of the present invention is monoclonal antibody capableof binding to an epitope of human melanoma-associated proteoglycan thatis not recognized by antibody 9.2.27. Also included are continuoushybrid cell lines capable of producing such antibodies.

These and other aspects of the invention will become apparent uponreference to the following description and attached drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the specificity of monoclonal antibodies generated tomelanoma alpha-2 macroglobulin when tested against three differentsources of alpha-2 macroglobulin in an ELISA. FIG. 1A depicts reactivitywith alpha-2 macroglobulin from melanoma spent culture medium; FIG. 1Bdepicts reactivity with purified alpha-2 macroglobulin from normal humanserum; and FIG. 1C depicts reactivity with bovine alpha-2 macroglobulin.One group (Group I) of monoclonal antibodies generated using melanomaalpha-2 macroglobulin reacted with alpha-2 macroglobulin obtained fromall three sources. Another group (Group II) reacted only with themelanoma and normal human serum forms of alpha-2 macroglobulin. A thirdgroup (Group III) reacted only with the melanoma form of alpha-2macroglobulin.

FIG. 2 depicts flow cytometry of monoclonal antibody toalpha-2-macroglobulin. Top panels indicate CEM (human T cell) or FMX(human melanoma) cells incubated with monoclonal antibodies reactivewith alpha-2-macroglobulin (alpha 2M) or intermediate filament antigens(anti-IFA), and stained with FITC-goat anti-mouse immunoglobulin. Bottompanels indicate FMX and CEM cells permeabilized with lysolecithin priorto incubation with alpha 2M or Anti-IFA. Anti-IFA is a positive controlfor intracellular, non-cell surface antigens.

FIG. 3 shows flow cytometry of melanoma-reactive monoclonal antibodiestested against (A) proteoglycan--positive and (B) proteoglycan-negativeA375 melanoma cells.

FIG. 3A illustrates binding of five anti-melanoma antibodies (9.2.27,NR-ML-02, NR-ML-03, NR-ML-04 and MR-ML-05) to A375 M/Mproteoglycan-positive cells. As shown in FIG. 3B, these same fiveanti-melanoma antibodies (9.2.27, NR-ML-02, NR-ML-03, NR-ML-04 andMR-ML-05) do not bind to A375 1° proteoglycan-negative cells.

FIG. 4 depicts trypsin-sensitivity of epitopes recognized byanti-proteoglycan monoclonal antibodies. 9.2.27 (◯); NR-ML-03 (□);NR-ML-04 ( ); NR-ML-05 ( ); and anti-HLA W6/32 (Δ).

BEST MODE FOR CARRYING OUT THE INVENTION

The method of the present invention for enhanced production ofantibodies circumvents prior art antigen purification steps, and alsoincreases the number of antigen-reactive hybridomas over that obtainedby prior art methodology, thus increasing the overall efficiency ofgenerating monoclonal and polyclonal antibodies to a desired antigen.

In order to carry out this invention, a relative amount of the desiredantigen within the antigen mixture needs to be determined. Thus, cellsurface extraction is carried out with agents such as lithiumdiiodosalicylate, butanol, isotonic urea, non-ionic detergents, or ionicdetergents to create a soluble extract. For example, non-detergentextractions are often used for preparation of peripheral membraneproteins. The soluble extract is an enriched source of antigen, when thetarget antigen represents 1% (w/w) or more of the extract preparation.However, less enriched extract preparations can also be utilized.

The extract preparation is then assayed for protein content. 100 ng to10 μg of protein in phosphate-buffered saline is added to polyvinylmicrotiter plates in 100 μl volumes. Following drying overnight, thepresence of the desired antigen is then detected by binding ofpolyclonal antisera or monoclonal antibody to the solid-phase targetantigen by ELISA techniques. For comparison, similar extracts fromnon-antigen-bearing cells can be utilized. The amount of antigen can beexpressed in titers by dilution of the target protein in wells. Havingestablished the presence of the desired antigen in the extractpreparation, one would titer the antibody or antibody sources versus thesolid-phase antigen. An antiserum or monoclonal antibody for preparationof the immunogen would be selected on the basis of highest titer,production in an appropriate species of animal, and/or subclass ofmurine immunoglobulin capable of binding to protein A-Sepharose®.

Typical protein concentrations used for forming insolubilized immunecomplexes are: 20 μg of polyclonal antibody per 20 μl of proteinA-Sepharose® (packed volume). Alternatively, 5-50 μg of MAbimmunoglobulin can be insolubilized on the same volume of proteinA-Sepharose®. An immunosorbent is formed by absorption, which is carriedout at room temperature for 2 hours, with end-over-end rotation,followed by washing in phosphate-buffered saline (PBS).

In an alternative embodiment, the immunosorbent is made by linking thedesired antibody directly to CNBr-Sepharose®. In yet another embodiment,the desired antibody is immobilized using anti-immunoglobulin-coatedmethacrylate beads or protein A-methacrylate beads.

Certain characteristics of the inert, immobilizing matrix may influencethe selection of a particular matrix for a particular purpose. Forinstance, while protein A-Sepharose®-bound immune complexes may besuitable for intraspenic or intraperitoneal injection, the size of theprotein A-Sepharose® bead renders such complexes impractical forintravenous administration in mice. Immune complexes insolubilized withanti-immunoglobulin-coated methacrylate beads or protein A-methacrylatebeads are better suited to murine intravenous administration, becausethe methacrylate beads are smaller in size than Sepharose®. It would beevident to one skilled in the art of immunology that a variety of inert,insoluble matrices having antibody-binding capabilities would besuitable for use within the present invention.

The immunosorbent is then utilized to absorb the antigen extract. Inorder to minimize the nonspecific adherence of extract proteins to theimmunosorbent, the extract is preabsorbed with normalimmunoglobulin-protein A-Sepharose®. Normal immunoglobulin can beobtained from rabbit or goat serum, or from a nonspecific mousemonoclonal antibody of the same subclass as the one used for absorptionof the specific antigen. Preabsorptions are carried out in three steps:(1) incubation of the nonspecific immunosorbent and extract preparationat room temperature for 2 hours; (2) transfer of the extract preparationto an aliquot of fresh nonspecific immunosorbent for an additional 2hours; and (3) transfer of the supernatant to a third aliquot ofnonspecific immunosorbent, with incubation overnight at 4° C. Followingthis depletion of nonspecific binding components, the antigenpreparation is retitered with antiserum or MAb to the specific antigen,and can be frozen in aliquots for subsequent preparation of theinsolubilized immune complex.

In vivo stability of the insolubilized immune complex is enhanced wherethe antibody and protein A are cross-linked by treatment with theN-succinimidyl ester of 4-azidosalicylic acid (NHS-ASA) (Tae et al.,Anal. Biochem. 121: 286, 1982). Alternatively, the antibody and antigencan be cross-linked by treatment with NHSASA. Cross-linking will preventdissociation in sera, thus increasing the in vivo retention of theimmune complex.

The amount of total protein added to the insolubilized antibody isapproximated on the basis of relative enrichment of the given antigen asassessed by ELISA. Thus, if the antigen preparation is a purifiedantigen, then 250 ng or less of pure antigen is utilized with eachimmunosorbent and each immunization. If the appropriate antigenconstitutes only 1% of the preparation, then typically 5-10 μg of theantigen preparation is utilized. If the soluble extract employed as thesource of antigen is a heterogeneous antigen mixture containing aconcentration of the given antigen which is less than 1%, then 10 μg ormore of the extract is utilized for absorption to the insolubilizedantibody. Following incubation at 4° C. for 2-14 hours, theinsolubilized immune complex is then washed with PBS and injected intothe peritonea of BALB/c mice.

If intrasplenic immunization is desired, the immunosorbent ishomogenized in a Dounce apparatus to produce smaller Sepharose®particles before intrasplenic injection with a 27-gauge needle. Whileintraperitoneal immunizations are scheduled weekly for 3-6 weeks,typically only one intrasplenic injection of 5-10 μg of the antigenpreparation is required. For both intraperitoneal and intrasplenicimmunizations, spleen cells are harvested 3-7 days after the finalimmunization, and hybridomas are produced by standard techniques.Hybridoma screenings are done by ELISA against the original antigentarget mixture used for immunization, as well as against target antigenmixtures known to not contain the antigen of interest.

Insolubilized immune complexes may also improve the immunogenicity ofpoorly immunogenic soluble antigens, thus allowing production ofhigh-titer antiserum or specific monoclonal antibodies which werepreviously unattainable. For instance, trichothecenes represent aspecies of mycotoxins produced by soil fungi of the class Fungiimperfecti or isolated from Baccharus megapotamica. Trichothecenes areextremely potent cytotoxins that act at the ribosomal level to inhibiteither initiation, elongation or termination of protein synthesis. Insome respects, trichothecenes resemble microbial or plant toxins, but incontrast to other "natural toxins", trichothecenes are very smallmolecules (i.e., 400-600 daltons). While the small size oftrichothecenes offers certain advantages for therapeutic applications,that same size combined with potent cytotoxic properties rendersgeneration of anti-trichothecene antibodies difficult, if notimpossible.

Within the present invention, anti-trichothecene antibodies aregenerated by immunizing experimental animals with insolubilizedtrichothecene-targeting protein conjugates. The targeting proteindisplays minimal or no reactivity with normal tissues. A preferredtargeting protein is a monoclonal antibody that is characterized asrelatively anti-tumor specific.

To further illustrate this embodiment of the invention, a targetingprotein is covalently linked to one or more trichothecene molecules. Inorder to further enhance immunogenicity, the conjugate is presented ininsolubilized form (i.e., on protein A-Sepharose® oranti-immunoglobulin-coated beads). When injected into a recipient, evenif some dissociation of insolubilized conjugate or insolubilized immunecomplex occurs in vivo, the released targeting protein-trichotheceneconjugate would not bind to normal cells. Instead, any releasedtargeting protein-trichothecene conjugate would be cleared by therecipient's foreign protein clearance mechanisms (assuming the targetingprotein is "foreign" to the recipient). Thus, by immunologicallypresenting a trichothecene in insolubilized conjugate or insolubilizedimmune complex form, the toxicity of the trichothecene component can begreatly reduced or eliminated. In addition, the targeting proteincomponent of the conjugate may function as a carrier for the"hapten-like" small trichothecene (<600 daltons), increasingtrichothecene immunogenicity.

When antigen is incorporated into an insolubilized immune complexthrough binding of antigen to a known monoclonal antibody, the epitoperecognized by the monoclonal antibody is presumably effectively masked.Immunization with antigen in insolubilized immune complexes with amasked epitope permits more efficient production of second generationmonoclonal antibodies, which are directed against non-masked epitopes ofthe antigen. This technique is useful for generating monoclonalantibodies directed against tumor-associated epitopes of non-tumor-associated antigens. Monoclonal antibodies which recognize differentepitopes of a given antigen might also be useful in differentiatingoncogene and proto-oncogene products.

For production of monoclonal anti-idiotypic antibodies, a human or mousemonoclonal antibody is absorbed to protein A-Sepharose®. The resultantimmunosorbent is washed free of unbound antibody and is then used toimmunize rodents. The hybridomas produced by fusion of immunized spleencells and myeloma cells are screened by one of two methods. If rats areimmunized with an insolubilized murine antibody on protein A-Sepharose®,then hybridoma screening is done by an indirect ELISA using anti-ratperoxidase-conjugated reagents. If mice are immunized, then hybridomascreening is done by inhibition of binding of enzyme-labeled specificantibody (idiotype) to target cells (antigen) by putative anti-idiotypicantibodies.

The success of insolubilized immune complex immunogens could be due to anumber of factors. The first is the relative enrichment of a givenantigen by absorption to specific antibody. However, this does notsuggest a rationale for the success of experiments utilizing polyclonalantisera as a source of immobilizing antibody. With polyclonal antisera,one would not expect this method to yield a great enrichment of a givenantigen. A second factor in this success could be the adjuvanicity ofinsolubilized protein A, which may play a role in enhancing B-celldifferentiation and proliferation. Soluble protein A is a well-knownmitogen and differentiation agent for mouse B-cells. Protein A could actas a secondary proliferation signal and cause clonal expansion ofB-cells presensitized to the target antigen. A third factor in thesuccess of these immunogens might be that immune complexes may be anatural mode of regulation of immune responses. Many studies haveindicated that antigen-antibody complexes traffic to follicular areas oflymph nodes and are bound preferentially to Fc receptors. Concentrationand subsequent presentation by macrophages can lead to sensitization ofB lymphoid cells, and thus to an enhancement of immune responsiveness.It would be expected that in vivo, insolubilized immune complexes aregradually released due to competition for protein A binding with thehost's immunoglobulin.

EXAMPLE 1 Generation of monoclonal antibodies utilizing monospecificpolyclonal antiserum

Alpha-2 macroglobulin (α₂ m) has been shown to be synthesized by humanmelanoma cells and secreted into spent culture medium (Morgan, JNCI72:7, 1984). The identification of this molecule was performed usingmonospecific polyclonal antisera raised to the normal serum form of α₂m. The melanoma cell form of α₂ m was shown to be of approximately thesame molecular weight, and to have the same subunit composition as,serum α₂ m. Monoclonal antibodies were then generated to the melanomacell form of alpha-2 macroglobulin utilizing an insoluble complex of thepolyclonal antisera to serum α₂ m and spent culture medium containingthe melanoma form of α₂ m. The specificity of these anti-α₂ m MAbs isshown in FIG. 1. Data from three groups of antibodies are shown. Thefirst group reacted with alpha-2 macroglobulin from normal serum,melanoma spent medium, and bovine serum. The second group of MAbrecognized only the human forms of alpha-2 macroglobulin in melanomaspent medium and in normal serum. The third group of antibodies reactedonly with the melanoma cell form of α₂ m, and did not recognize eitherthe normal human or bovine serum forms. These results demonstrate thatmonoclonal antibody production can be enhanced by utilizing monospecificpolyclonal immune complexes, and that the monoclonal antibodiesgenerated can be selective for a tumor cell form of an antigen ratherthan for its normal serum counterpart, even though the tumor cell formis molecularly very similar to the normal serum antigen.

The specificity of anti-α₂ m monoclonal antibodies was furthercharacterized by flow cytometry. A human melanoma cell line thatsynthesizes α₂ m (FMX) was incubated with anti-α₂ m MAb. In order todetect α₂ m that is synthesized, but not expressed on the cell surface,FMX cells were also permeabilized with lysolecithin prior to incubationwith MAb. Intact and permeabilized human T cells (CEM) that lack α₂ mserved as a negative control. Anti-intermediate filament MAb acted as apositive control for access to intracellular antigens afterpermeabilization. FIG. 2 demonstrates binding of anti-α₂ m MAb tointra-cellular α₂ m only, indicating that α₂ m is not expressed on thesurface of FMX cells. CEM cells were consistently nonreactive,demonstrating that endocytosis of bovine α₂ m from the culture mediumdid not interfere with the assay.

EXAMPLE 2 Generation of monoclonal antibodies to transforming growthfactor using polyspecific polyclonal antibody and a heterogeneousantigen preparation

Alpha transforming growth factor (αTGF) may be important in the controlof tumor growth, and in particular, in endocrine growth regulation.Monoclonal antibodies against αTGF have been difficult to generate, eventhough synthetic peptides are available which represent all or part ofthis peptide growth factor's amino acid sequence. Polyspecific rabbitantiserum raised against whole melanoma cells was titrated against apreparation TGF isolated by reverse-phase HPLC from melanoma urine. Theantiserum displayed a low titer (1:4) versus the α TGF preparation,which was approximately 20% αTGF and 80% other contaminating urinaryproteins by SDS-polyacrylamide gel electrophoresis. Thisantiserumantigen preparation was bound to protein A-Sepharose®, andmonoclonal antibodies were generated utilizing six weekly immunizations.Examples of hybridoma clones derived from these fusions are shown inTable 1.

                  TABLE 1                                                         ______________________________________                                        MONOCLONAL ANTIBODIES REACTIVE WITH                                           ALPHA TRANSFORMING GROWTH FACTOR                                              Reactivity Pattern*                                                                           Percent of Clones                                             ______________________________________                                        TGF.sup.+, EGF.sup.+                                                                          23                                                            TGF.sup.-, EGF.sup.+                                                                          69                                                            TGF.sup.+, EGF.sup.-                                                                          8                                                             ______________________________________                                         *Monoclonal antibodies were assayed by solidphase ELISA for binding to        urinary αTGF from melanoma patients and to purified human EFG.     

Most of the monoclonal antibodies generated in response to the urinaryαTGF preparation reacted with human epidermal growth factor (EGF) aswell as with αTGF. Some monoclonal antibodies raised to urinary αTGFreacted preferentially with EGF, but not with a 17-amino acid peptidederived from the C-terminus of αTGF. One monoclonal antibody displayedspecificity for αTGF and was nonreactive with human EGF. These resultsdemonstrate that a non-immunogenic peptide growth factor such as αTGFcan be made immunogenic by insolubilization via an immune complex.

EXAMPLE 3 Generation of monoclonal anti-idiotypic antibodies to humanIgM

Monoclonal anti-idiotypic antibodies to human IgM present on the cellsurface of B-cell lymphomas may be useful as therapeutic agents in thetreatment of some forms of B-cell lymphoma. In order to speed up thegeneration of these antibodies, and in order to use only smallquantities of a sometimes limiting IgM immunogen, an intrasplenicimmunization approach was tested. Soluble human IgM injectedintrasplenically was not an effective immunogen for generatinghybridomas. However, as shown in Table 2, when the human IgM wascomplexed to protein A-Sepharose® via murine monoclonal antibody tohuman IgM, the IgM became immunogenic, resulting in about 1% of theclones being directed to the antigen combining site of the human IgMmyeloma protein.

                                      TABLE 2                                     __________________________________________________________________________    ASSESSMENT OF INTRASPLENIC IMMUNIZATION FOR THE PRODUCTION OF ANTI-           IDIOTYPIC HYBRIDOMAS                                                                        Time Period Between Immunization and Fusion                                   3 Days             7 Days                                       Immunogen     ID.sup.a                                                                           ID-PAS.sup.b                                                                        ID-MAb-PAS.sup.c                                                                      ID   ID-PAS                                                                              ID-MAb-PAS                        __________________________________________________________________________    No. of wells seeded                                                                         1380 552   552     1380 920   920                               Hybrid efficiency (%)                                                                       7.5  98    98      0    40    40                                Wells containing secreted                                                                   0    159                                                                              (29)                                                                             210 (38)                                                                              0    16 (1.7)                                                                            56  (6.0)                         murine immunoglobulin (%)                                                     Wells containing secreted                                                                   0    3  (0.5)                                                                            5   (0.9)                                                                             0    3  (0.3)                                                                            11  (1.2)                         idiotypic hybridomas.sup.d (%)                                                Wells demonstrating                                                                         0    0     5   (0.9)                                                                             0    13 (1.4)                                                                            6   (0.7)                         anti-IgM reactivity                                                           __________________________________________________________________________     All fusions were performed with the mouse myeloma cell line 8.653 at a        fusion ratio of 4:1 spleen:myeloma cells. Following fusion, cells were        distributed into 96well plates so that approximately 2 × 10.sup.6       cells were seeded into every well.                                            .sup.a Human IgM (idiotype) immunoglobulin only.                              .sup.b Human IgM (idiotype) immunoglobulin complexed to protein ASepharos      ®.                                                                       .sup.c Human IgM (idiotype) immunoglobulin complexed to protein ASepharos     ® via a mouse monoclonal antihuman IgM bridge.                            .sup.d Antiidiotype hybridomacontaining wells were assayed by solid phase     ELISA for binding to a pool of five human IgM paraproteins or to the IgM      used for immunization.                                                   

The specificity of these anti-IgM idiotypic monoclonal anti-bodies wasthen examined. The murine monoclonal antibodies were assayed for bindingto a panel of human IgM paraproteins and individual IgM preparations.Table 3 demonstrates that these monoclonal antibodies bound only toautologous IgM, but not to other IgM preparations, thus showing likelyanti-idiotypic specificity.

                                      TABLE 3                                     __________________________________________________________________________    SELECTIVITY OF MURINE MONOCLONAL ANTI-IDIOTYPIC ANTIBODIES AS DETERMINED      BY ELISA*                                                                              Monoclonal Antibodies Generated by Immunization with Patient                  IgM                                                                                                           Anti-Patient                                  Anti-Patient HM     Anti-Patient GD                                                                           LH       Anti-Patient KT             Screening                                                                              HM-1                                                                              HM-2                                                                              HM-3                                                                              HM-4                                                                              HM-5                                                                              GD-1                                                                              GD-2                                                                              GD-3                                                                              LH-1                                                                              LH-2 KT-1                                                                              KT-2                                                                              KT-3                Immunoglobulin                                                                         3.sup.b                                                                           μ                                                                              2a  2b  1   1   1   μ                                                                              1   1    2a  1   1                   __________________________________________________________________________    HM (IgM) .sup. 1.00.sup.c                                                                  0.88                                                                              1.00                                                                              1.10                                                                              1.30                                                                              0.00                                                                              0.01                                                                              0.00                                                                              0.00                                                                              0.00 0.00                                                                              0.00                                                                              0.00                GD (IgM) 0.00                                                                              0.06                                                                              0.00                                                                              0.00                                                                              0.00                                                                              1.12                                                                              1.00                                                                              1.10                                                                              0.00                                                                              0.00 0.00                                                                              0.00                                                                              0.02                LH (IgM) 0.02                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.01                                                                              0.01                                                                              0.00                                                                              1.32                                                                              1.18 0.00                                                                              0.00                                                                              0.00                KT (IgM) 0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.01                                                                              0.00                                                                              0.00 0.35                                                                              0.98                                                                              1.07                DM (IgM) 0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.01 0.00                                                                              0.00                                                                              0.00                Meloy (IgM).sup.d                                                                      0.03                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.02 0.01                                                                              0.00                                                                              0.02                Cappel (IgM).sup.d                                                                     0.00                                                                              0.00                                                                              0.01                                                                              0.05                                                                              0.00                                                                              0.01                                                                              0.03                                                                              0.00                                                                              0.00                                                                              0.00 0.01                                                                              0.01                                                                              0.02                NHS.sup.e                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00                                                                              0.00 0.00                                                                              0.00                                                                              0.00                __________________________________________________________________________     Anti-idiotypes generated by the indicated patients' IgM were assayed by       solid phase ELISA for binding to a series of IgM paraproteins. Results ar     expressed as the OD.sub.405.                                                  .sup.a Antiidiotype monoclonal antibody designation.                          .sup.b Immunoglobulin isotype and subclass of the antiidiotype monoclonal     antibody.                                                                     .sup.c Positive ELISA results are underlined.                                 .sup.d Pools of human IgM myeloma proteins; Meloy5 donors, Cappel12           donors.                                                                       .sup.e Normal human serum.                                               

EXAMPLE 4

Generation of rat monoclonal anti-idiotypic antibodies againstmonoclonal antibody which recognizes a tumor-associated antigen of humanmelanoma cells

Antibody 9.2.27, which recognizes a human melanoma-associated antigen (a250 kd glycoprotein/proteoglycan complex, also referred to as"proteoglycan antigen"), is useful in tar-geting human melanoma cells inpatients (Schroff et al., JNCI 74: 299, 1985; Oldham et al., J. Clin.Oncol. 2:1235, 1984). The antigen recognized by 9.2.27 represents adifficult biochemical purification task, because it requires selectiveextraction techniques combined with CsCl gradient centrifugation andantibody affinity chromatography. The antigen has only been isolated insmall quantities, which have been insufficient for immunization.Insufficient quantities of antigen also limit the potential use of the250 kd component as a vaccine. However, generation of monoclonalanti-idiotypic antibodies that recognize MAb 9.2.27 could provide asubstitute vaccine. A number of immunization methodologies were assayed,both in rats and mice, to determine the optimal method for inducingmurine monoclonal anti-idiotypic antibodies to a murine mono-clonalantibody. These methods included a traditional immunization scheduleemploying soluble MAb together with Freund's adjuvant. Immunogens werealso created by conjugating the MAb to an albumin carrier, withimmunization across species barriers (into rats and hamsters) to enhanceimmunogenicity. None of these methodologies were successful ingenerating anti-idiotypic antibodies.

A protocol according to the present invention, utilizing MAb 9.2.27insolubilized on protein A-Sepharose®, was successful in generatinganti-idiotypic antibodies in rats. The results are shown in Table 4.

The rat monoclonal antibodies were shown to bind to the immunizing MAb9.2.27, but not to the same subclass or other subclasses of mousemonoclonal antibodies. Thus, insolubilizing a murine MAb via an immunecomplex may aid in decreasing self-tolerance to mouse monoclonalantibodies in mice or rats.

                                      TABLE 4                                     __________________________________________________________________________    SPECIFICITY OF RAT ANTI-9.2.27 IDIOTYPIC ANTIBODIES*                          Test   Monoclonal Rat Anti-Murine Idiotypic Antibodies                        Antibody                                                                             9131A                                                                             3A11G5                                                                             3A11F5                                                                              9129                                                                              85-25-3A                                                                           85-25-3B                                       __________________________________________________________________________    Control                                                                              0.000                                                                             0.003                                                                              0.003 0.004                                                                             0.002                                                                              0.007                                          MOPC   0.010                                                                             0.028                                                                              0.025 0.033                                                                             0.013                                                                              0.014                                          21 (G1).sup.a                                                                 D.sub.3 (G1)                                                                         0.097                                                                             0.173                                                                              0.151 0.209                                                                             ND   0.014                                          9-2-27 1.052                                                                             >2.0 >2.0  >2.0                                                                              0.167                                                                              1.477                                          (G2a)                                                                         RPC5   0.074                                                                             0.095                                                                              0.085 0.130                                                                             0.024                                                                              0.018                                          (G2a)                                                                         D.sub.3 (G2a)                                                                        0.112                                                                             0.120                                                                              0.133 0.187                                                                             0.008                                                                              0.004                                          T101   0.077                                                                             0.117                                                                              0.110 0.160                                                                             0.015                                                                              ND                                             (G2a)                                                                         ZME (G2a)                                                                            0.096                                                                             0.115                                                                              0.103 0.230                                                                             0.016                                                                              0.017                                          MOPC   0.022                                                                             0.028                                                                              0.022 0.033                                                                             0.012                                                                              0.006                                          141 (G2b)                                                                     MOPC   0.000                                                                             0.004                                                                              0.006 0.008                                                                             0.008                                                                              0.005                                          195 (G2b)                                                                     FLOPC  0.000                                                                             0.017                                                                              0.015 0.024                                                                             0.010                                                                              0.003                                          21 (G3)                                                                       Y5606  0.052                                                                             0.000                                                                              0.000 0.000                                                                             0.010                                                                              0.003                                          (G3)                                                                          MOPC   0.000                                                                             0.000                                                                              0.003 0.006                                                                             0.002                                                                              0.018                                          104E (M)                                                                      TEPC   0.000                                                                             0.035                                                                              0.036 0.054                                                                             0.010                                                                              0.000                                          183 (M)                                                                       TEPC   0.000                                                                             0.053                                                                              0.049 0.066                                                                             ND   ND                                             15 (1)                                                                        __________________________________________________________________________     *Murine monoclonal antibody 9.2.27, which recognizes a human                  melanomaassociated antigen, was used as an immunogen in rats. The             resultant rat antimurine idiotype monoclonal antibodies were assayed by       solid phase ELISA for binding to murine monoclonal antibodies or myeloma      proteins, as indicated. A mouse antirat peroxidase conjugate was used in      the solid phase ELISA.                                                        .sup.a Immunoglobulin isotype and subclass corresponding to each test         monoclonal antibody or myeloma protein are indicated in parentheses.     

EXAMPLE 5 Generation of multiple monoclonal antibodies directed againsta variety of epitopes of a human melanoma-associated proteoglycanantigen

Insolubilized immune complexes were employed in immunization strategiesdesigned to generate a large number of monoclonal antibodies to a humanmelanoma-associated proteoglycan antigen.

Three types of immunogens were used for generating antibodies to theproteoglycan antigen. The first was whole cells. The cell lines used forthis purpose included A375 M/M, SK MEL-28, SK MEL-31 grown in DMEM withten percent fetal bovine serum (J.R. Scientific, Woodland, Calif.) andA375 M/M grown in serum-free media (Iscove's medium with 5 mg/literinsulin and transferrin and 5 μg/liter selenium). Immunizations wereonce a week for three weeks, with 1×10⁶ cells subcutaneously. A secondimmunogen was prepared from extracts of melanoma cells (e.g., FMX or SKMEL-28 absorbed to lentil-lectin- or wheat germ agglutinin-Sepharose 4B®(Morgan et al., Hybridoma 3:233, 1984)). A third type of immunogenutilized insolubilized immune complexes (e.g., proteinA-Sepharose®-bound complexes of 9.2.27 and melanoma NP-40 extracts(Giardina et al., J. Immunol. Meth., 89:1-7, 1986)). The second andthird type of immunogens were injected once a week for six consecutiveweeks before fusion. A fourth immunization schedule combined a wholecell priming dose with immune complex immunization (e.g., complex ofbeads covalently attached to goat anti-mouse IgG, 9.2.27 and melanomaNP-40 extracts), in which mice were immunized with a three injections ofwhole cells followed by two injections with the insolubilized immunecomplex.

Hybridomas were produced by the fusion of immunized spleen cells withP3/X63-Ag8-653 myeloma cells at a 10:1 spleen:myeloma ratio. Thehybridomas were first examined for binding to melanoma cells, B cells,and T cells using an indirect two-stage immunoperoxidase procedure(Woodhouse et al., J. Natl. Canc. Inst. 74:383, 1985). Hybridomaspositive for binding to melanoma cells, but negative for binding to Band T cells, were subsequently screened by flow cytometry.

Briefly, single cell suspensions of B and T cells and of A375 M/Mproteoglycan-positive and A375 1° proteoglycan-negative melanoma cellswere incubated with monoclonal antibodies at a concentration of 1 μg ofantibody per 1×10⁶ cells. Fluorescein isothiocyanate-conjugated goatanti-mouse antibody (Tago Inc., Burlingame, CA) was then used to detectbound monoclonal antibody. Target cell binding was analyzed on a Epics Ccytofluorograph (Coulter Diagnostics, Hialeah, FL) using standardizedfluorescein particles (Becton Dickinson, Mountain View, Calif.) tocalculate free fluorescein equivalents. FIG. 3 shows the resultsobtained with MAb 9.2.27 and four melanoma-positive monoclonalantibodies that react with proteoglycan-positive cells (FIG. 3A), but donot bind to proteoglycan-negative cells (FIG. 3B).

Monoclonal antibodies reactive with the proteoglycan-positive melanomacell line, but not with the normal or antigen-negative cell lines, werethen examined in an indirect ELISA assay (Morgan and Mclntyre, Canc.Res. 43:3155, 1983). The assay was performed with NP40 extracts ofmelanoma cells. A number of putative anti-proteoglycan monoclonalantibodies reacted with the melanoma cell extracts in a manner similarto MAb 9.2.27, while some did not react.

The monoclonal antibodies that produced ELISA patterns similar to MAb9.2.27 were then further evaluated by an immunoperoxidase stainingprocedure using samples of human melanoma, skin and kidney. The lattertwo normal tissues represent all of the known normal tissue crossreactivities of 9.2.27. Monoclonal antibodies that intensely stainedmelanoma specimens, basal cells within epidermis of skin and collectingtubules in kidney were considered to be putative anti-proteoglycanantibodies ("9.2.27-like") and were investigated further.

A double determinant ELISA (two-site immunoassay) was performed toassess reactivity to proteoglycan. The double determinant ELISA wasutilized in two formats. The first format utilized MAb 9.2.27 F(ab')₂ ascapture antibody, with the addition of a standardized source of antigen,SK MEL-28 NP40 extract, followed by the addition of supernatants ofputative anti-proteoglycan antibodies. Following incubation, boundmonoclonal antibody was detected with a biotin anti-mouse Fc-specificreagent. Antibodies which gave positive color development in this assaywere considered anti-proteoglycan antibodies. In a second format of theassay, the antigen in the extract was captured by solid-phase antibody,which included MAbs 9.2.27, NR-ML-02, NR-ML-03, NR-ML-04 and NR-ML-05.The same antibodies, biotinylated, were used in various combinations todetect bound antigen. Antibodies recognizing the same epitope underthese conditions gave no color development, whereas antibodiesrecognizing distinctive epitopes gave significant color development.This assay assumes that only a single epitope per antibody is present onthe antigen, which is tested by having both capture and detection withthe same antibody.

Antibodies still showing reactivity at this level were evaluated byimmunoprecipitation and SDS-PAGE to confirm the reactivity to theproteoglycan antigen. Five anti-proteoglycan antibodies were chosen formore detailed evaluation.

A summary of the number of fusions and the number of putativeanti-proteoglycan antibodies is shown in Table 5.

                  TABLE 5                                                         ______________________________________                                        SUMMARY OF FUSIONS AND RESULTS                                                            Number   Number of Number of Putative                             Type of     of       Hybrids   Anti-Proteoglycan                              Immunogen   Fusions  Screened  MAbs                                           ______________________________________                                        Lectin/Extract                                                                            7        1,619     1                                              Whole Cells 7        1,767     4                                              Immune Complex                                                                            5        1,261     0                                              Whole Cells plus                                                                          2        1,302     67                                             Immune Complex                                                                ______________________________________                                    

Whole cell immunizations gave rise to relatively few putativeanti-proteoglycan antibodies. However, the combination of sensitizationwith whole cells followed by boosting with the insolubilized immunecomplex of MAb 9.2.27 and proteoglycan antigen resulted in a largenumber of putative anti-proteoglycan antibodies. Five putativeanti-proteoglycan monoclonal antibodies were selected for furtheranalysis. These antibodies and the cell lines that produce them areshown in Table 6.

Deposits of the designated cell lines were made to the A.T.C.C.,Rockville, Md., and were assigned the designated accession numbers.

                  TABLE 6                                                         ______________________________________                                        MONOCLONAL ANTIBODY AND HYBRID CELL LINE                                      DESIGNATIONS                                                                  Monoclonal Antibody                                                                        Hybrid Cell Line                                                                           ATCC Accession No.                                  ______________________________________                                        NR-ML-02     NR-ML-02 A1923                                                                             HB-9352                                             NR-ML-03     NR-ML-03 A2215                                                                             HB-9349                                             NR-ML-04     NR-ML-04 A1993                                                                             HB-9351                                             NR-ML-05     NR-ML-05 A2106                                                                             HB-9350                                             NR-ML-06     NR-ML-06 A3026                                                                             HB-9348                                             ______________________________________                                    

The reactivity profiles of these antibodies are presented in Table 7.

                  TABLE 7                                                         ______________________________________                                        REACTIVITY OF ANTI-MELANOMA ANTIBODIES BY                                     ELISA                                                                                    Absorbance at 415 nm Against Target                                                     NR-     NR-   NR-   NR-                                  ELISA TARGET 9.2.27  ML-02   ML-03 ML-04 ML-05                                ______________________________________                                        Pooled B&T   0.047   0.053   0.071 0.065 0.027                                cell                                                                          cultures                                                                      Pooled       0.771   0.575   0.660 0.571 0.908                                melanoma                                                                      cultures                                                                      A375 met     1.097   0.886   0.961 0.688 1.036                                mix melanoma                                                                  cells                                                                         A375 primary 0.007   0.001   0.018 0.018 0.051                                melanoma                                                                      cells                                                                         A375 met     1.008   0.772   0.808 0.774 1.130                                mix NP-40                                                                     extract                                                                       SK-MEL-31    0.953   0.645   0.598 0.587 0.869                                NP-40                                                                         extract                                                                       ______________________________________                                    

Detailed immunoperoxidase evaluation of the anti-proteoglycan monoclonalantibodies was performed. The patterns of reactivity were identical toMAb 9.2.27, including a low degree of antigenic heterogeneity asevidenced by intense and homogeneous staining of representative melanomasections, with all the antibodies staining virtually all of the tumorcells. Smooth muscle and endothelial cell reactivity was also found withall of the antibodies.

Epitope comparisons of the anti-proteoglycan monoclonal antibodies werealso performed, using both double determinant and addition assays, inELISA and flow cytometry formats, respectively. Table 8 indicates thatMAbs 9.2.27, NR-ML-03 and NR-ML-05 did not give a positive signal whenused for both capture and detection, suggesting that each recognizes aunique epitope. In contrast, MAbs NR-ML-02 and NR-ML-04 did not give apositive signal in any combination, and thus appear to recognize thesame epitope.

                  TABLE 8                                                         ______________________________________                                        EPITOPE COMPARISONS BY DOUBLE DETERMINANT                                     SOLID-PHASE ELISA                                                             Biotinylated                                                                           Absorbance with Indicated Capture Antibody                           Detector         NR-ML-   NR-ML- NR-ML- NR-ML-                                Antibody 9.2.27  02       03     04     05                                    ______________________________________                                        9.2.27   0.016   1.657    1.554  1.558  1.167                                 NR-ML-02 1.731   0.107    1.229  0.094  0.927                                 NR-ML-03 0.636   0.527    0.054  0.575  0.406                                 NR-ML-04 1.847   0.030    1.445  0.119  1.078                                 NR-ML-05 0.928   0.880    0.901  0.740  0.121                                 ______________________________________                                         Antigen sourcemelanoma patient ascites fluid                             

This hypothesis was confirmed by additive cell surface binding with flowcytometry. Using the fluorescence intensity of each antibody, Table 9shows that combinations of antibodies recognizing distinct epitopes gave100% or more of the theoretical sum of the fluorescence intensity of thetwo antibodies alone minus that of the control antibody. NR-ML-04 andNR-ML-02 gave only one-half the expected value, indicating that theseMAbs recognize the same epitope. Sixty-one other anti-proteoglycanmonoclonal antibodies were screened for additivity to the fourdeterminants recognized by 9.2.27, NR-ML-03, NR-ML-05 andNR-ML-02/NR-ML-04. None of the 61 antibodies examined recognized thesame epitope as MAb 9.2.27, the antibody used in the immune compleximmunization scheme. Ten of the sixty-one antibodies appeared torecognize the same determinant as NR-ML-02 and NR-ML-04, whereas onlythree antibodies recognized the same determinants as NR-ML-03 orNR-ML-05. Forty-eight of the antibodies did not recognize an epitopeidentified by the five characterized antibodies.

                  TABLE 9                                                         ______________________________________                                        EPITOPE COMPARISONS BY INDIRECT                                               IMMUNOFLUORESCENCE                                                                       KD            ZME   NR-   NR-   NR-                                9.2.27     200    P97    08    ML-02 ML-04 ML-05                              ______________________________________                                        9.2.27 96                                                                     KD200  96      118                                                            P97    92      107    119                                                     ZME08  86       58     95  99                                                 NR-Ml- 90      100    105  91    97                                           02                                                                            NR-Ml- 88      104    109  92    59                                           04                                                                            NR-Ml- 95       94    100  97    85    72    104                              05                                                                            NR-Ml- 100     N/D    N/D  N/D   N/D   N/D   N/D                              06                                                                            ______________________________________                                         (Percent of theoretical staining intensity on cultured melanoma cells; N/     = not done)                                                              

Representative anti-proteoglycan monoclonal antibodies were thenevaluated for Fc-mediated binding using THP-1, a continuous monocyticcell line that expresses Fc receptors for murine and human IgG, as shownin Table 10. Since the THP-1 cell line does not express receptors forIgG-1, binding of the IgG-1 anti- proteoglycan monoclonal antibodies(NR-ML-02 and NR-ML-04) was at background levels. Interestingly, theIgG-2b NR-ML-05 antibody also did not bind, whereas both IgG-2aantibodies, NR-ML-03 and 9.2.27, bound extremely well.

                  TABLE 10                                                        ______________________________________                                        Fc RECEPTOR BINDING COMPARISON BY FLOW                                        CYTOMETRY                                                                                           Fluorescein Test: Control                               Antibody      Isotype Equivalents Ratio                                       ______________________________________                                        Secondary Alone                                                                              --     37,000      1.0                                         NR-ML-02      IgG1    43,000      1.2                                         NR-ML-04      IgG1    61,000      1.6                                         NR-ML-05      IgG2b   55,000      1.5                                         NR-ML-03      IgG2a   186,000     5.0                                         9.2.27        IgG2a   196,000     5.3                                         ______________________________________                                    

The proteoglycan epitope recognized by MAb 9.2.27 exhibitstrypsin-sensitivity (A. C. Morgan, Jr. et al., Hybridoma 1:27, 1981).Because trypsin-sensitivity may relate to relative exposure of anepitope at a target cell membrane, epitopes of the anti-proteoglycanantibodies generated herein were examined for trypsin-sensitivity.

FMX cells were exposed to varying concentrations of trypsin, and bindingof monoclonal antibodies was evaluated by flow cytometry and expressedas percent fluorescence intensity of untreated FMX cells. FIG. 4 showstwo classes of trypsin-sensitive epitopes. The first, epitomized by MAb9.2.27, was highly trypsin-sensitive, requiring only low levels oftrypsin to reduce antibody binding. This was in contrast to the relativetrypsin-resistance of the HLA-A,B,C determinant recognized by MAb W6/32and the epitope recognized by NR-ML-05. Trypsin-resistant epitopes maybe located relatively close to the plasma membrane. Of 46 differentanti-proteoglycan monoclonal antibodies examined, twenty recognizedepitopes that were trypsin-sensitive.

Radiolabeled NR-ML-02, NR-ML-05 and 9.2.27 were further evaluated bybiodistribution and tumor uptake in nude mice xenografts. Table 11presents tumor localization of monovalent fragments of 9.2.27, NR-ML-02and NR-ML-05.

                  TABLE 11                                                        ______________________________________                                        TUMOR LOCALIZATION OF ANTI-PROTEOGLYCAN                                       ANTIBODIES ASSESSED BY BIODISTRIBUTION IN                                     NUDE MICE CARRYING MELANOMA XENOGRAFTS                                                Type of  Number   Average   Average                                           Frag-    of Deter-                                                                              Tumor     Tumor:                                    Antibody                                                                              ment     minations                                                                              % Dose/Gram                                                                             Blood                                     ______________________________________                                        9.2.27  Fab      16       2.08 ± 0.55                                                                          10.83 ± 2.87                           NR-ML-02                                                                              Fab'      8       2.05 ± 0.51                                                                          3.79 ± 1.31                            NR-ML-05                                                                              Fab      113      2.75 ± 0.74                                                                          23.96 ± 16.83                          ______________________________________                                    

Papain digestion of 9.2.27 and NR-ML-05 produced Fab fragments; NR-ML-02Fab' fragments were produced by pepsin digestion followed by cysteinereduction. Monovalent fragments were labeled with technetium-99m using apreformed diamido-dimercapto ligand system (European Patent Application0188256) and injected intravenously at a dose of 50 mg per mouse. At 20hours post-injection the mice were sacrificed, appropriate tissues wereremoved, weighed and counted, and data were corrected for isotopicdecay.

Tumor uptake, as assessed by percent of injected dose per gram of tumor,was not significantly different for either NR-ML-02 or NR-ML-05 comparedwith 9.2.27 by a t-test (p>0.05). While 9.2.27 had a higher tumor:bloodratio than NR-ML-02 (p<0.01), the tumor:blood ratio for NR-ML-05 washigher than for either of the other two monoclonal antibodies (p<0.01).

The higher tumor uptake of NR-ML-05 cannot be attributed to antibodyaffinity, since the affinity of NR-ML-05 Fab is lower than the affinityof 9.2.27 Fab. In addition, 9.2.27 recognizes a greater number ofbinding sites on cultured cells than NR-ML-05. However, the tumor uptakeof NR-ML-05 may correlate with proteolytic digestion within necroticregions of the tumor, since NR-ML-05 is relatively trypsin-resistant.

EXAMPLE 6 Production of second generation monoclonal antibodies usingNR-LU-10 - antigen immune complexes

Insolubilized immune complexes were employed in immunization protocolsdesigned to generate "NR-LU-10-like" monoclonal antibodies.

Briefly, two BALB/c mice were injected intraperitoneally with 10⁷ LS-174colon carcinoma cells on days 1, 7, 14, 21 and 28. The mice wereinjected intravenously with insolubilized immune complexes on days 35,44 and 51. Insolubilized immune complexes were formed by combining 200μl of a 1% solution of goat anti-mouse IgG beads (Kirkegaard & PerryLaboratories, Inc., Gaithersburg, MD), 5 mg NR-LU-10 and LS-174 NP-40extract (about 100 μg protein). The mice were sacrificed on day 54, andthe spleens were harvested and frozen in liquid nitrogen.

In a first fusion performed with the frozen spleen cells, hybridomaswere produced by the fusion of 7×10⁷ immunized spleen cells and 1.5×10⁷P3/X63-Ag8-653 myeloma cells (5:1 spleen:myeloma). The resultinghybridomas were examined by ELISA techniques for binding to LS-174 NP-40extract and to antigen captured by NR-LU-10. Hybridomas positive forbinding to both LS-174 NP-40 extract and to NR-LU-10-captured antigen,or to NR-LU-10-captured antigen only, were further cloned and retestedin the same ELISA. This fusion generated two hybridoma clones thatsecreted IgG3 antibodies that were positive in the two-site immunoassay.By immunohistology, these monoclonal antibodies displayed a tissuedistribution similar to that of NR-LU-10. The hybridoma clones weredesignated A5792 and A5793 (secreting NR-LU-02 and NR-LU-03 monoclonalantibodies, respectively), and have been assigned ATCC Accession Nos. HB10193 and HB 10194, respectively.

In a second fusion performed with the same frozen spleen cells,hybridomas were produced by the fusion of 1.7×10⁸ immunized spleen cellsand 2.3×10⁷ P3/X63-Ag8-653 myeloma cells (7:1 spleen:myeloma). Whentested as described for the first fusion, the second fusion generatedone hybridoma clone producing a "NR-LU-10-like" IgG₃ antibody, asdetermined by two-site immunoassay and immunohistology. This hybridomaclone was designated A5791 (secreting NR-LU-01 monoclonal antibody), andhas been assigned ATCC Accession No. HB 10192.

Another immunization protocol included intraperitoneal injection of twoBALB/c mice with 250 μl packed volume of an LS-174(NP-40) extract boundto Concanavalin A agarose on days 1, 7, 12 and 21. On days 28, 35 and42, the mice were intravenously injected with insolubilized immunecomplexes formed by combining 200 μl of a 1% solution of goat anti-mouseIgG methacrylate beads, 5 mg NR-LU-10 and LS-174(NP-40) extract (about100 μg protein). The mice were sacrificed on day 45, the spleensremoved, and 10⁸ spleen cells were fused with 2×10⁷ P3/X63-Ag8-653myeloma cells. One hybridoma clone, designated NR-LU-05, was identifiedthat was positive in the two-site immunoassay and showed a similartissue distribution to NR-LU-10 by immunohistology.

Each of the above-described fusion protocols generated additionalhybridoma clones that were positive in the two-site immunoassay, butwhich showed a tissue specificity different from that of NR-LU-10 byimmunohistology. These monoclonal antibodies may recognize epitopes ofthe antigen which are shared with other, more widely represented tissueantigens.

EXAMPLE 7 Production of monoclonal antibodies directed against acytotoxic trichothecene

Insolubilized immune complexes were employed in an immunization protocoldesigned to generate anti-verrucarin A monoclonal antibodies.

Verucarin A is a member of a group of small molecules (i.e., 400-600daltons) called trichothecenes, which are a species of mycotoxinsproduced by soil fungi of the class Fungi imperfecti or isolated fromBaccharus megapotamica. Trichothecenes are extremely potent cytotoxinsthat act at the ribosomal level to inhibit either initiation, elongationor termination of protein synthesis.

In order to generate antibodies to verrucarin A (VA), the toxicity ofthe molecule must be greatly reduced or eliminated. Further, due to thesmall size of VA (502 daltons), VA requires association with a carrierto be an effective immunogen. Conjugation of VA and targeting proteinprovides an avenue whereby the cytotoxicity of VA is decreased while itsimmunogenicity is increased.

Briefly, two BALB/c mice were injected intrasplenically on days 1 and 11with 1 μg VA-monoclonal antibody conjugate that was insolubilized using100 μl of a 1% solution of goat anti-mouse IgG methacrylate beads. Themonoclonal antibody, NR-ML-05, was a murine IgG_(2b) directed against amelanoma-associated proteoglycan. The mice were sacrificed on day 14 andthe spleens were harvested.

Hybridomas were produced by the fusion of about 1×10⁸ immunized spleencells (obtained from combined spleens) and 2×10⁷ P3/X63-Ag8-653 myelomacells (5:1 spleen:myeloma). The hybridomas were screened by ELISA forbinding to a human serum albumin (HSA)-VA conjugate and to HSA alone. Of30 wells displaying growth, two hybridoma clones produced antibodyreactive with HSA-VA, but not with HSA alone. To confirm that thesehybrids recognized VA and not a "neo-determinant" expressed on the VAconjugate, hybridoma supernatants were tested for reactivity with twoantibody-VA conjugates produced using different chemical linkages, andwere found to be positive against both. Furthermore, free VA was shownto inhibit binding of both monoclonal antibodies to HSA-VA. Thesemonoclonal antibodies, both of the IgM class, were designated NR-Va-01and NR-Va-02.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notto be limited except as by the appended claims.

What is claimed is:
 1. A method for producing a monoclonal antibodyspecific for an epitope distinct from that recognized by an existingmonoclonal antibody, comprising the steps of:absorbing a firstmonoclonal antibody directed against a first epitope of a polyvalentantigen onto an inert, insoluble matrix capable of bindingimmunoglobulin, thereby forming an immunosorbent; combining saidimmunosorbent with said polyvalent antigen, forming an insolubilizedimmune complex wherein said first epitope is masked by said firstmonoclonal antibody; immunizing an animal with said insolubilized immunecomplex; fusing spleen cells from said immunized animal to myeloma cellsto form a hybridoma capable of producing a second monoclonal antibodydirected against a second epitope of said polyvalent antigen; culturingsaid hybridoma to produce said second monoclonal antibody; andcollecting said second monoclonal antibody as a product of saidhybridoma.
 2. The method of claim 1 wherein said inert, insoluble matrixcapable of binding immunoglobulin is selected from the group consistingof CNBr-activated agarose, CNBr-activated Sepharose®, protein A-agarose,protein A-Sepharose®, protein A-methacrylate beads, andanti-immunoglobulin-coated methacrylate beads.
 3. The method of claim 1,comprising, after the step of absorbing the antibody, the additionalstep of crosslinking the antibody to the inert, insoluble matrix.
 4. Themethod of claim 1, comprising, after the step of combining the antigen,the additional step of crosslinking the antibody and antigen.
 5. Themethod of claim 1 wherein said antigen is a component of a heterogeneousmixture.
 6. The method of claim 5 wherein said heterogeneous mixture isselected from the group consisting of human melanoma cell extract, humancolon carcinoma extract, human breast carcinoma extract, and human lungcarcinoma extract.
 7. The method of claim 1 wherein said first epitopeis a tumor-associated epitope recognized by monoclonal antibodyNR-LU-10.
 8. The method of claim 1 wherein the antigen is atumor-associated antigen.
 9. The method of claim 1 further comprising,after the step of culturing, the step of screening hybridoma culturemedium to determine the presence of said second monoclonal antibody. 10.The method of claim 9 wherein the screening step is performed by doubledeterminant ELISA.
 11. The cell line NR-LU-01 A5791.
 12. A monoclonalantibody produced by the cell line of claim
 11. 13. The cell lineNR-LU-02 A5792.
 14. A monoclonal antibody produced by the cell line ofclaim
 13. 15. The cell line NR-LU-03 A5793.
 16. A monoclonal antibodyproduced by the cell line of claim 15.